Process for recovering L-phenylalanine

Abstract: An industrial process for recovering L-phenylalanine from a side stream obtained from the production of α-APM at high yield is established. For recovering L-phenylalanine from at least one side stream generated in the process for the production of α-APM, L-aspartic acid or an aqueous solution thereof is added when the hydrolysis of the above side stream is performed under acidic conditions with a mineral acid, or when the pH of the reaction mixture obtained by hydrolysis is adjusted to 5 to 6 to precipitate L-phenylalanine.

Description: This invention relates to a process for producing α-L-aspartyl-L-phenylalanine methyl ester (hereinafter referred to as α-APM), which is about 200 times as sweet as sucrose and is in great demand for dietary sweeteners due to its good sweet taste and low caloric value. More particularly, this invention relates to a process for recovering L-phenylalanine from the side stream generated from a process for producing α-APM. Description of Prior Art Various processes for producing α-APM are known, for example a process which comprises reacting N-protected L-aspartic anhydride with L-phenylalanine methyl ester (US Patent Ser. No. 3786039), a process which comprises reacting N-protected L-aspartic anhydride with L-phenylalanine (US Patent Ser.No. 4173562), a process which comprises condensing N-benzyloxycarbonyl-L-aspartic acid and L-phenylalanine methyl ester with an enzyme (Japanese Patent Application Laid-open No. 92729-1978). However, to employ them as low cost industrial process, it is necessary to recover and reuse L-phenylalanine and L-aspartic acid from side streams obtained during a process for the production of α-APM such as α-APM mother liquor or α-APM·HCl mother liquor, since these side streams contain a significant amount of L-phenylalanine derivatives, L-aspartic acid derivatives and β-L-aspartyl-L-phenylalanine methyl ester and α-APM. Of these, it is very important to efficiently recover L-phenylalanine, which is an expensive amino acid. For recovering L-phenylalanine from a side stream generated during a process for the production of α-APM a process disclosed in Japanese Patent Application Laid-open No. 97812-1973 is known. The process comprises the hydrolysis of a solution containing β-L-aspartyl-L-phenylalanine methyl ester in the presence of a mineral acid, adjusting the pH of the reaction solution to 6 for the crystallization and separation of L-phenylalanine. The mother liquor after separation still contains a considerable amount of L-phenylalanine. Accordingly, it is expected that the recovery may be further improved if the solubility of L-phenylalanine could be reduced. EP-A-55612 also relates to the recovery of L-phenylalanine. Problem to be solved by the Invention The problem to be solved by the invention is to establish an industrial process to efficiently recover L-phenylalanine from a side stream generated in the production of α-APM. Means to solve the Problem The present inventors have intensively studied a process for the efficient recovery of L-phenylalanine from a side stream obtained in a process for the production of α-APM. As the result, it was unexpectedly found that solubility of L-phenylalanine in the aqueous solution is reduced and the recovery of L-phenylalanine is improved by adding L-aspartic acid or an aqueous solution thereof when the pH of the reaction mixture obtained by the hydrolysis of said side stream in the presence of a mineral acid is adjusted to 5 - 6 in order to precipitate L-phenylalanine. The addition of L-aspartic acid or an aqueous solution thereof may take place during or after the hydrolysis step and before the step of pH adjustment. The present invention will be illustrated in detail. Typical side streams obtained in the production of α-APM, which are the object of the present invention, include, for example, (i) a mother liquor obtained by crystallization and separation of α-APM from an aqueous medium, which is obtained by condensing N-benzyloxycarbonyl-L-aspartic anhydride and L-phenylalanine methyl ester, and by deprotecting upon reduction (US Patent Ser. No. 3786039); (ii) a mother liquor obtained by separating α-APM·HCl precipitated upon condensation of N-formyl-L-aspartic acid anhydride and L-phenylalanine methyl ester, followed by treatment with methanol and hydrochloric acid at high concentration (US Patent Ser.No. 4684745); (iii) a mother liquor obtained after separation of α-APM·HCl precipitated upon esterification of α-AP with methanol and hydrochloric acid in an aqueous medium (US Patent Ser.No. 4173562). The mineral acid used in the present invention includes hydrochloric, sulfuric, phosphoric acid and the like. Hydrochloric acid is desirable considering the easy removal of its salts generated by pH adjustment after hydrolysis. During hydrolysis, the concentration of the acid is preferably 1 - 12 N, particularly 2 - 6 N. The amount used is desirably more than 1, particularly 1.5 to 10 mole per 1 mole of amino acid and amino acid residue contained in said side stream. The temperature for hydrolysis is desirably 60 to 120°C, especially 90 to 110°C. The reaction time can not be limited because it varies depending on various factors. Too long a reaction time promotes racemization. Generally sufficient is a reaction time up to 20 hours. The pH of thus obtained hydrolyzate is adjusted with an alkali such as sodium hydroxide. L-Aspartic acid or an aqueous solution thereof may be added either before or after such pH adjustment. The addition may of course be carried out before hydrolysis without any problems. The amount of L-aspartic acid to be added is not limited. A sufficient amount is not less than 10 % (by mole) based on that of L-aspartic acid present in the hydrolyzate. The pH is selected from 5 to 6 from the viewpoint of the isoelectric point of L-phenylalanine. The alkali used for pH adjustment includes sodium hydroxide, potassium hydroxide, sodium carbonate, ammonia and the like. Sodium hydroxide is most preferable from the viewpoint of cost. The alkali may be directly added. However, it is desirably added in the form of an aqueous solution for easiness of pH adjustment. The temperature for pH adjustment is not particularly limted. However, in order to precipitate L-phenylalanine, dissolved after pH adjustment, as much as possible, it is required to mature the mixture at a temperature not higher than 40°C for an hour or more. The aqueous solution of L-aspartic acid to be added may be prepared by dissolving crystalline L-aspartic acid in water. However, it is efficient to use an aqueous solution containing L-aspartic acid obtained from the production of α-APM. One embodiment is the above mother liquor obtained from crystallization and separation of L-phenylalanine. This mother liquor contains L-aspartic acid, which is generated by hydrolysis in addition to L-phenylalanine. Generally, in the process for the production of α-APM, the pH of the mother liquor obtained by separating L-phenylalanine is adjusted to 2 to 3 which is the isoelectric point of L-aspartic acid, and the precipitating L-aspartic acid is separated and recovered from the mother liquor to be reused. However, there remains L-aspartic acid dissolved in this mother liquor. Usually, this mother liquor is discarded as a waste solution. Accordingly, it is efficient to use the mother liquor as the above aqueous solution. Further, not only L-aspartic acid but also L-phenylalanine exists dissolved in the mother liquor. Therefore the total recovery of L-phenylalanine is further improved if the mother liquor is utilized as an aqueous solution of L-aspartic acid thereby recovering L-phenylalanine in the mother liquor. This mother liquor may be directly used. However, if it is directly used, inorganic salt may be precipitated upon increased concentration during precipitation of L-phenylalanine since there exists inorganic salt in the mother liquor (equimolar to the mineral acid used for hydrolysis). Accordingly, it is preferred to previously desalt the mother liquor. The desalting ratio may vary depending on various factors so that it can not be specified. Generally, it is sufficient within the range of 20 to 90%. For desalting, this mother liquor is concentrated and the precipitated inorganic salts are removed by filtration. Alternatively, a method utilizing loose reverse osmotic membrane at an appropriate pH can be employed. L-Aspartic acid used in the present invention may be easily recovered by the following method. That is, after the objective L-phenylalanine is separated, the pH of the filtrate is adjusted to 2 to 3, and the precipitated crystals are isolated and recovered with L-aspartic acid generated by hydrolysis. The present invention will be explained in detail in the following examples. Example 1 A mother liquor of α-APM·HCl (0.9 l) (concentration of HCl is 3.5 N) was hydrolyzed by heating and refluxing at 110°C for 6 hours. The analysis by an amino acid analyzer showed that the reaction solution contained 112.0 g of L-aspartic acid and 116.5 g of L-phenylalanine. After 56.0 g of L-aspartic acid was added to the reaction solution, pH was adjusted to 5.6 with 48% aqueous solution of caustic soda at 70 °C. The pH adjusted solution was cooled to 10 °C and stirred for 3 hours. The precipitated crystal was collected by filtration and analyzed by an amino acid analyzer. As the result, the content of L-phenylalanine was 109.3 g, yield was 93.8% (based on L-phenylalanine in the hydrolyzate). On the other hand, the concentration of L-phenylalanine in the filtered mother liquor was 0.56 g/dl. Comparative Example 1 The reaction solution, hydrolyzed in the same manner as in Example 1, was adjusted to pH 5.6 with 48% aqueous solution of caustic soda at 70 °C and subsequently cooled to 10 °C and stirred for 3 hours. The precipitated crystal was collected by filtration and analyzed by an amino acid analyzer. As the result, the content of L-phenylalanine was 103.1 g, yield was 88.5% (based on L-phenylalanine in the hydrolyzate). On the other hand, the concentration of L-phenylalanine in the filtered mother liquor was 1.06 g/dl. Example 2 35% Hydrochloric acid (85 g) was added to a mother liquor of α-APM·HCl (15.0L) and concentrated to 0.9 L under reduced pressure. 35% Hydrochloric acid (concentration of HCl, 4.0 N) was added to the concentrate, then hydrolysis was carried out at 110 °C for 6 hours. Analysis by an amino acid analyzer showed that the reaction solution contained 105.8 g of L-aspartic acid and 112.6 g of L-phenylalanine. After 105.8 g of L-aspartic acid was added to the reaction solution, pH was adjusted to 5.6 with 48% aqueous solution of caustic soda at 50 °C. The pH adjusted solution was cooled to 10 °C and stirred for 3 hours. The precipitated crystal was collected by filtration and analyzed by an amino acid analyzer. As the result, the content of L-phenylalanine was 102.8 g, yield was 91.3% (based on L-phenylalanine in the hydrolyzate). Comparative Example 2 L-Phenylalanine, obtained in the same manner as in Example 2, except that amino acid was added to the hydrolyszte, was 96.3 g, yield was 85.5% (based on L-phenylalanine in the hydrolyzate). Example 3 A mother liquor of α-APM·HCl (118 l) was hydrolyzed and adjusted to pH 5.6. The precipitated L-phenylalanine was removed by filtration. The resulting mother liquor is adjusted to pH 2.8, and the precipitated L-aspartic acid was removed by filtration. The resulting mother liquor (190 l) (concentration of ingredients: L-phenylalanine, 0.9 g/dl; L-aspartic acid, 1.7 g/dl; sodium chloride, 25.7 g/dl) was adjusted to pH 5.1 with 48 % caustic soda. After dilution by the 1.4 fold amount of water, the product was desalted and concentrated using loose reverse osmotic membrane at 30°C. As loose reverse osmotic membrane, NTR-7250 (manufactured by NITTO DENKO CORPORATION, membrane area, 1.8 m 2 ) was used. The above mother liquor was passed through the loose reverse osmotic membrane under a pressure of 27 bar (Kgf/cm 2 ) to concentrate to 2.2 times, simultaneously removing 50 % of sodium chloride in the mother liquor, transferring into the permeated solution. Subsequently, water was added to the concentrate to dilute to the 2.1 fold, and again concentrated using loose reverse osmotic membrane under a pressue of 27 bar (Kgf/cm 2 )(concentration ratio, 2.6 fold), and finally, 100 l of concentrated and desalted solution (content: L-phenylalanine, 1.3 g/dl; L-aspartic acid, 3.1 g/dl; sodium chloride, 11.9 g/dl) was obtained by removing 75 % of sodium chloride contained in the original mother liquor, transferring it into the permeated solution. To the mother liquor of α-APM·HCl (186 l, same as that used in Example 1; containing 25.5 kg of L-phenylalanine, 24.2 kg of L-aspartic acid; HCl concentration, 3.5N) were added 100 l of the above concentrated and desalted solution (containing 3.1 kg of L-aspartic acid and 1.3 kg of L-phenylalanine). The resulting solution was concentrated under reduced pressure to obtain 177 l of a concentrate. The concentrate was heated and refluxed at 110°C for 5 hours for hydrolysis. The reaction solution was adjusted to pH 7.0 with 48 % caustic soda at 70°C, then cooled to 10°C and stirred at the same temperature for 3 hours. The precipitated crystal was collected by filtration and analyzed by an amino acid analyzer. As the result, the content of L-phenylalanine was 24.3 kg, yield was 96.4 % (based on L-phenylalanine in the mother liquor of α-APM·HCl). Comparative Example 3 Water (100 l) was added to a mother liquor of α-APM·HCl (186 l), concentrated under reduced pressure to obtain concentrate (177 l). The concentrate was hydrolyzed by heating and refluxing at 110°C for 5 hours. The reaction solution was adjusted to pH 5.6 with 48 % caustic soda at 70°C, then cooled to 10°C and stirred at that temperature for 3 hours. The precipitated crystal was collected by filtration and analyzed by an amino acid analyzer. As the result, the content of L-phenylalanine was 21.6 kg, yield was 85.7 % (based on L-phenylalanine in the mother liquor of α-APM·HCl). Example 4 After α-APM·HCl mother liquor (118 l) was hydrolyzed, the pH was adjusted to 5.6 and the precipitated L-phenylalanine was removed by filtration. Further the pH of the resulting mother liquor was adjusted to 2.8 and the precipitated L-aspartic acid was removed by filtration.The resulting mother liquor (190 l) (content: L-phenylalanine, 0.9 g/dl; L-aspartic acid, 1.7 g/dl; sodium chloride, 25.7 g/dl) was adjusted to pH 5.1 with 48 % caustic soda. The solution was concentrated to 110 l under reduced pressure, and then stirred at 60°C for an hour. The precipitated crystal of sodium chloride was removed by filtration. The filtrate (88 l, containing 3.2 kg of L-aspartic acid and 1.7 kg of L-phenylalanine) was added to α-APM·HCl mother liquor (186 l, containing 24.2 kg of L-aspartic acid and 25.2 kg of L-phenylalanine) and concentrated to 177 l under reduced pressure. The concentrate was hydrolyzed by heating and refluxing at 110°C for 5 hours, then the pH was adjusted to 5.6 with 48 % caustic soda at 60°C. Subsequently, the solution was cooled to 10°C and stirred at the temperature for 3 hours. The precipitatd crystal was collected by filtration and analyzed by an amino acid analyzer. As the result, the content of L-phenylalanine was 24.9 kg, the yield was 98.8 % (based on L-phenylalanine in the α-APM·HCl mother liquor). Advantages of the Invention L-Phenylalanine can be recovered at high yield by adding L-aspartic acid or an aqueous solution thereof for the precipitation of L-phenylalanine.

Claims: A process for recovering L-phenylalanine from at least one of the side streams obtained in the production of α-L-aspartyl-L-phenylalanine methyl ester, which comprises subjecting the side stream to a hydrolysis step in the presence of a mineral acid and thereafter adjusting the pH to 5 to 6, wherein L-aspartic acid or an aqueous solution thereof is added before, during or after the hydrolysis step and before the step of pH adjustment for precipitation of L-phenylalanine. A process according to claim 1, wherein the aqueous solution of L-aspartic acid to be added is a mother liquor obtained after separation of said precipitated L-phenylalanine or a solution with reduced mineral salt content upon desalting of the mother liquor. A process according to claim 1, wherein the aqueous solution of L-aspartic acid to be added is a mother liquor obtained by separating said precipitated L-phenylalanine, adjusting pH of said mother liquor to 2 to 3 followed by separating the precipitated L-aspartic acid, or a solution with reduced mineral salt upon desalting of the mother liquor. A process according to any of the claims 1 to 3, wherein the mineral acid is hydrochloric acid. A process according to any of the claims 2 to 4, wherein loose reverse osmotic membrane is employed for desalting. A process according to any of the claims 2 to 4, wherein said desalting operation comprises precipitating and filtering mineral salt after concentration.

Inventors: KATAOKA TAKEHIKO, TAKEMOTO TADASHI, KATAOKA, TAKEHIKO, TAKEMOTO, TADASHI, KATAOKA, TAKEHIKO C/O CENTRAL RESEARCH LAB., TAKEMOTO, TADASHI C/O CENTRAL RESEARCH LAB.
Applicants: AJINOMOTO KK, AJINOMOTO CO., INC.
Application year: 1994
Main classification: C07C227
IPC codes: C07C229, C07K1, C07K5, C07C227